Microfluidic array devices and methods of manufacture thereof

ABSTRACT

Microfluidic nozzle array devices are provided with the body of each device having at least one nozzle extending outwardly from one surface of the body. The microfluidic nozzle array devices are fabricated using an injection molding process and find particular utility in a wide range of applications, including but not limited to nanospray/electrospray applications, mass spectrometer applications, optical spectrometry applications, spotting applications (i.e., DNA or protein array), etc. Manufactured nozzles can include sealed nozzle apexes which are subsequently opened by focusing energy from an energy source on the sealed apex resulting in the opening of the nozzle channel.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/627,640, filed Nov. 12, 2004, related to applicant'sU.S. Pat. No. 6,800,809, which are hereby incorporated by reference inits entirety.

TECHNICAL FIELD

The present invention relates to microfluidic devices, and moreparticularly, to microfluidic array devices that can be used to deliverone or more samples through one or more nozzles that are formed as partof the microfluidic array device. Exemplary manufacturing methods forfabrication of the microfluidic array devices are also disclosed as wellas exemplary uses for the microfluidic array devices. For example, themicrofluidic array device is suitable for operations designed forlab-on-a-chip functions including analysis of components in the samplefluid by means of optical spectrometry, mass spectrometry, etc.

BACKGROUND

There has been a growing interest in the development and manufacturingof microscale fluid systems for the acquisition of chemical andbiochemical information and as a result of this effort, microfluidics isconsidered an enabling technology for providing low cost, highversatility devices to operations, such as combinatorial chemistry fordrug lead discovery and large-scale protein profiling to name a few.Generally, a microfluidic device (which is also often referred to as alab-on-a-chip device) is a planar device having one or more micron sizedchannels formed therein and can also include reservoirs, valves, flowswitches, etc. The microfluidic features are designed to carry outcomplex laboratory functions, such as DNA sequencing.

In the absence of using microfluidic devices, the above processes andothers are carried out in a manner that is very time intensive and thus,costly. For example, large-scale protein profiling is commonly carriedout laboriously but pervasively in the biotechnological andpharmaceutical industries. One particular application of microfluidicdevices is to provide microfluidic channels that represent the means toseparate analytes in a mixture using techniques, such as capillaryelectrophoresis and liquid chromatography.

Microfluidic devices have traditionally been fabricated fromsubstantially planar substrates with microfabrication techniques thathave been borrowed from the electronics industry, such asphotolithography, chemical etching, and laser ablation techniques. Whenconstructing the microfluidic devices in this manner, the microfluidicchannels that are formed lie parallel to the surface of one planarsurface of the substrate, and the channel is sealed by bonding a secondplanar substrate to the planar substrate containing the channel. Thetechniques for detecting materials, such as analytes, that are disposedin the microfluidic channels have for the most part been mainly opticaltechniques. Fluid transport in the microfluidic devices traditionallyentails using electroosmotic, electrokinetic and/or pressure-drivenmotions of liquid and particles as the means for fluidly transportingsuch materials.

While the stacking of multiple layers of planar substrates to form amicrofluidic structure having layered microfluidic channels is possiblein terms of its fabrication, the prevailing detection technology(optically based detection technology) limits the practicality offabricating such a structure since parallel operation of multiple layersof the planar substrates containing multiple microfluidic separationchannels is not practical due to each microfluidic separation channelrequiring its own light source and detector.

One detection technology that is fast becoming the detection techniqueof choice in the biotechnology and pharmaceutical industries is massspectrometry (MS). Mass spectrometry provides more chemical informationabout the material being tested (e.g., analytes) than other singledetection techniques. For example, molecular weight and even chemicalcomposition of the analytes from small drug candidate molecules to largeprotein molecules can be successfully analyzed by mass spectrometry (MS)and its related technique that is referred to as MS-MS. In MS-MS, amolecule is ionized and analyzed for molecular weight in the first stageof the mass spectrometer, and then the same molecular ion, called the“parent”, is fragmented inside the mass spectrometer to produce“daughter” ions that are further analyzed to give the chemicalcomposition of the parent molecule.

While some progress has been made to interface microfluidic devices witha mass spectrometer, there are still several shortcomings that must beovercome in order to make this interfacing process more practical. Forexample, one technique that has been discussed involves drilling a smallhole, large enough to accommodate a glass or quartz capillary, into theend of the microfluidic channel that is formed by glass substrates and aglass or quartz capillary is then inserted into the drilled hole to actas a nozzle for electrospray ionization. This approach is laborious andis impractical for high throughput operations where many such holes haveto be drilled sequentially into the substrates.

In another technique that has been disclosed, a protrusion termed“electropipette” extends from the edge of the substantially planarsubstrate. The microfluidic channel in this extended region is formed bytwo planar substrates as in the microfluidic channels that are formed inthe rest of the microfluidic device. The outside dimensions of the tipstructure include a thickness that is equal to the thickness of the twoplanar substrates. It has also been disclosed to fabricate an array ofnozzles using microfabrication techniques, such as deep ion reactiveetching on a silicon wafer. However, the use of silicon wafers as thesubstrates greatly limits the ability to individually activate eachnozzle because of the potential of dielectric breakdown caused by thehigh voltage applied to the nozzle to create the electrosprayconditions, and the volume behind the nozzle made by deep ion reactiveetching is extremely difficult to be accessed by conventional means ofliquid handling equipment. Integrating this silicon-based nozzle arrayto microfluidic devices, which are typically made of glass or polymers,is also extremely difficult. The cost of fabricating the nozzles onsilicon is also very high.

While injection molding has been discussed as a process for formingmicrofluidic devices, there are a number of limitations that haveequally been associated with such discussion of injection moldablemicrofluidic devices. For example, it has heretofore been discussed thatthere are limitations on what size dimensions can be formed when aninjection molding process is used to form the microfluidic features.Prior to the present applicant, there was a lack of appreciation andunderstanding that an injection molding process can be used to form amicrofluidic device having microfluidic features with dimensions lessthan 100 μm. As a result, the use of injection molding as a fabricationprocess was limited since many microfluidic applications require themicrofluidic device to have microfluidic features (e.g., channels) thathave dimensions less than 100 μm and more particularly, less than 50 μm.

It would therefore be desirable to provide microfluidic devices,especially microfluidic array devices incorporating nozzles, thatovercome the deficiencies of the traditional microfluidic devices andmore particularly, the deficiencies that are related to the techniquesfor fabricating these devices and also to the use of such devices.

SUMMARY OF THE INVENTION

The present application generally relates to microfluidic devices.According to one aspect, a microfluidic device is provided and includesa body having a first surface and an opposing second surface. At leastone channel is formed through the body such that the channel extendsfrom the first surface to the opposing second surface with the channelhaving an open reservoir section formed at the first surface. Themicrofluidic device further includes at least one nozzle that isdisposed along the second surface. The nozzle is in fluid communicationwith one channel such that each channel terminates in a nozzle openingthat is formed as part of the nozzle tip. Unlike traditionalmicrofluidic devices, the exemplary microfluidic device has one or morechannels that are open at each end and are formed substantiallyperpendicular to both the first surface and the second surface where thenozzle is formed.

According to another aspect, the nozzle is conically shaped with thechannel extending therethrough and terminating at the nozzle opening. Inone exemplary embodiment, the nozzle opening has a diameter equal to orless than 100 μm, preferably equal to or less than 50 μm and morepreferably, equal to or less than 20 μm; and an outside diameter of thenozzle, as measured at a tip portion thereof, is less than about 150 μmand preferably is equal to or less than about 100 μm, and morepreferably equal to or less than 50 μm. For electrospray typeapplications, a conductive region is formed on the nozzle, preferably ata tip portion thereof, to permit a voltage to be applied to the tipportion of the nozzle. As the sample fluid is discharged from thenozzle, the electric field that is created by the conductive regionserves to vaporize and ionize the sample and form a fine mist containingthe sample. This fine mist can then be injected into an inlet port of ananalytical instrument, such as a mass spectrometer, to detect andanalyze components of the sample and obtain certain information aboutthe components.

In another aspect of the present application, the microfluidic nozzlearray device is formed by an injection molding process that permits themicrofluidic nozzle array device to have the above dimensions. A mold isfirst fabricated with the mold being a negative impression of thechannel architecture and nozzle array that are formed as part of themicrofluidic nozzle array device. Preferably, the mold is made of ametal material and with at least some portions of the mold beingpolished to a high degree of finish, i.e., a mirror finish. Morespecifically, the polishing of a conical portion of the mold that isused to form the nozzle results in the nozzle having a very smooth outersurface and also facilitates the flow of an injected polymer within thisnozzle region, thereby increasing the accuracy and the efficiency of theinjection molding process. A suitable polymeric material is injectedinto the mold and is then cured to form the injection moldedmicrofluidic nozzle array device. After the device has sufficientlycooled, the microfluidic nozzle array device is then removed from themold.

The exemplary microfluidic nozzle array devices disclosed herein can beused in a number of different applications. For example, the device isparticularly well suited for operations designed for lab-on-a-chipfunctions including the detection of components in the sample fluid bymeans of UV, visible light and by means of mass spectrometry. Moreover,it will be appreciated that the microfluidic nozzle array device can beused in a wide range of other applications in which similar conventionalmicrofluidic devices have or could be used. For example, themicrofluidic nozzle array device can be used for spotting DNA or proteinarray on a substrate instead of using the conventional capillary wickingmethods that are now used. The microfluidic nozzle array device can alsobe used for spotting the plate for matrix-assisted laser desorptionionization (MALDI), replacing the pipette and capillary spottingmethods. In addition, the microfluidic nozzle array device can be inother spray or spotting type applications where it is desired to producea fine stream of sample fluid.

In yet another aspect of the present invention, the method offabricating the microfluidic nozzle array device includes the step offorming the nozzle opening and/or the nozzle channel using a laser. Morespecifically, the apex end of the nozzle channel may be sealed onpurpose or may be sealed during the manufacturing process and a laserbeam is focused on this sealed apex portion so as to eliminate thesealing material and open up the nozzle tip. Alternatively, themicrofluidic nozzle array device can be formed such that the bodyincluding the reservoir and a solid nozzle are formed and the channelthat extends through the solid nozzle to the reservoir is formed usingthe laser. In other words, the open nozzle channel is formed using thelaser such that a channel is formed that defines the nozzle tip openingand forms an entrance into the reservoir.

These and other features and advantages of the exemplary embodimentsdisclosed herein will be readily apparent from the following detaileddescription taken in conjunction with the accompanying drawings, whereinlike reference characters represent like elements.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The foregoing and other features of the exemplary embodiments will bemore readily apparent from the following detailed description anddrawings of illustrative embodiments that are not necessarily drawn toshow exact likeness or necessarily to scale in which:

FIG. 1 is a top perspective view of a microfluidic device having anarray of nozzles incorporated therein according to a first exemplaryembodiment;

FIG. 2 is a cross-sectional view taken along the line 2-2 of FIG. 1;

FIG. 3 is a top plan view of the microfluidic device according to FIG. 1illustrating placement of electrodes around the nozzles and theconnections between the electrodes and electrical contacts formed at oneedge of the microfluidic device;

FIG. 4 is a top perspective view of a microfluidic device having anarray of nozzles incorporated therein according to a second exemplaryembodiment;

FIG. 5 is a cross-sectional view of the microfluidic device according toFIG. 4;

FIG. 6 is a perspective view of an exemplary mold used to manufacturethe microfluidic device of FIG. 1;

FIG. 7 is a cross-sectional view of first and second dies in a closedposition that is used to manufacture the microfluidic device of FIG. 4;

FIG. 8 is a cross-sectional view of first and second dies of the moldillustrating another embodiment where a gap is formed between a pin ofthe first mold and a nozzle forming feature of the second mold;

FIG. 9 is a cross-sectional view illustrating a mold arrangement forfabricating a micron sized nozzle opening;

FIG. 10 is a top plan view of a tile arrangement formed of a number ofstrips connected to one another with each strip including a nozzlearray, wherein one of the strips is removed and placed in closeproximity to a mass spectrometer;

FIG. 11 is a cross-sectional view of one microfluidic channel/nozzlearrangement wherein a sample reservoir is sealed by a member having apolymeric cover sheet which is insertable and movable within thereservoir for discharging the sample through a nozzle opening;

FIG. 12 is a cross-sectional view of one microfluidic channel/nozzlearrangement wherein a sample reservoir is sealed by a member having anelastic sealing base which is insertable and movable within thereservoir for discharging the sample through a nozzle opening;

FIG. 13 is a cross-sectional view of one microfluidic channel/nozzlearrangement where a sample reservoir is sealed by a piston device havinga bore extending therethrough for injecting a fluid into the samplereservoir to cause the sample to be discharged through a nozzle opening;

FIG. 14 is a top plan view of an exemplary microfluidic nozzle arraydevice;

FIG. 15 is a cross-sectional view taken along the line 14-14;

FIG. 16 is a cross-sectional side elevational view illustrating themicrofluidic device of FIG. 5 being used in UV spectrophotometry;

FIG. 17 is a top plan view of a retaining base for releasably holding anumber of microfluidic nozzle subunit structures;

FIG. 18 is a cross-sectional view taken along the line 18-18 of FIG. 17;

FIG. 19 is a top plan view of a retaining base according to anotherembodiment for releasably holding a number of microfluidic nozzlesubunit structures;

FIG. 20 is a local cross-sectional view of a microfluidic device havinga nozzle portion that is sealed by a thin membrane that is subsequentlyremoved using a laser device;

FIG. 21 is a local cross-sectional view of the nozzle portion of FIG. 20with the thin membrane removed as a result of activation of the laser;

FIG. 22 is a local cross-sectional view of a microfluidic device havinga nozzle portion that is sealed by a thin membrane that is laterpartially removed using a laser device;

FIG. 23 is a local cross-sectional view of the nozzle portion of FIG. 22with the thin membrane partially removed as a result of activation ofthe laser;

FIG. 24 is a cross-sectional view of a sealed solid nozzle extendingfrom a microfluidic substrate base that has a reservoir formed therein;and

FIG. 25 is a cross-sectional view of the nozzle of FIG. 24 with amicrofluidic channel being formed through the nozzle as a result of anenergy source being focused thereon.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Referring first to FIGS. 1-2 in which an exemplary microfluidic device10 according to one embodiment is illustrated. The microfluidic device10 has a substrate body 20 that is formed of a polymeric material, aswill be described in greater detail hereinafter, and has at least onemicrofluidic channel 30 that is formed in the substrate body 20. Morespecifically, the substrate body 20 has a first surface 22 and anopposing second surface 24 with the microfluidic channel 30 being formedbetween the first and second surfaces 22, 24 such that the microfluidicchannel 30 extends the complete thickness of the substrate body 20. Themicrofluidic channel 30 is thus open at both a first end 32 at the firstsurface 22 and a second end 34 at the second surface 24. The second end34 of the microfluidic channel 30 is formed in a protrusion 50 that isformed on the second surface 24 of the substrate body 20. According toone exemplary embodiment, the protrusion 50 has a tapered shape (inwardtaper) such that it forms a generally conical structure with the opensecond end 34 preferably being formed at an apex of the conicalstructure. The tapered protrusion 50 serves as a nozzle that delivers asample (i.e., a liquid) that is loaded into the microfluidic device 10.

It will be appreciated that in contrast to traditional microfluidicdevices, the microfluidic channel 30 is formed in a perpendicular mannerin the substrate body 20 in that the microfluidic channel 30 ispreferably formed so that it is substantially perpendicular to the firstand second surfaces 22, 24 of the substrate body 20. As illustrated, apredetermined number of microfluidic channels 30 and nozzles 50 can beformed in one substrate body 20. The microfluidic channels 30 can bearranged according to any number of different patterns. For example andas illustrated in the exemplary embodiment of FIGS. 1 and 2, whichillustrate a preferred arrangement, a plurality of microfluidicchannels/nozzles are arranged in regular arrays having spacing that isidentical to or similar to spacing of microtiter plates. For example, if96 microfluidic channels/nozzles are desired, then the 96 microfluidicchannels/nozzles are arranged in an 8×12 grid with spacing of about 9 mmbetween each microfluidic channel/nozzle structure. For a 384 microtiterarray, the microfluidic channels/nozzles are placed in a 16×24 grid withspacing of about 4.5 mm. While not entirely to scale, FIG. 2 generallyillustrates a section of a microfluidic channel/nozzle array havingspacing of about 4.5 mm.

According to the present exemplary embodiments, each nozzle 50 isconstructed so that its dimensions are measured in microns. The specificconfigurations of the nozzle 50 and the microfluidic channel 30 are bestshown in FIG. 2. As illustrated, the first end 32 of the microfluidicchannel 30 is in the form of a reservoir 60 (i.e., an annular cavity)that tapers inwardly to an intermediate channel section 36. Theintermediate channel section 36 also has a tapered construction in thatit tapers inwardly toward the second end 34 and the nozzle 50 formed atthe second surface 24 of the substrate body 20. Thus, the dimensions ofthe microfluidic channel 30 are greatest at the first end 32, where thereservoir is formed, and are at a minimum at the second end 34 at a tipportion 52 of the nozzle 50. According to one exemplary embodiment, theopen second end 34 of the microfluidic channel 30 formed in nozzle 50has an inside diameter of about 100 μm or less, preferably equal to orless than 50 μm and more preferably, equal to or less than 20 μm; and anoutside diameter of the nozzle, as measured at a tip portion thereof, isless than about 150 μm and preferably is equal to or less than about 100μm, and more preferably equal to or less than 50 μm. The inside diameterof the microfluidic channel 30 opens gradually in a direction away fromthe nozzle 50 to about several hundred μm as the microfluidic channel 30traverses through the thickness of the substrate body 20 and eventuallythe microfluidic channel 30 is formed to a diameter of about 1 mm todefine the reservoir at the first end 32. The length of the microfluidicchannel 30 can be tailored to a given application depending upon anumber of factors, such as the desired volume of the reservoir definedat first end 32 and also the thickness of the substrate body 20.According to one exemplary embodiment, the microfluidic channel 30 has alength of about 3 mm or greater. However, the aforementioned dimensionsare merely recited to illustrate one exemplary embodiment and it will beunderstood that the microfluidic device 10 can be fabricated to haveother dimensions.

The volume of the reservoir 60 should be such that it can hold an amountof sample material that is typically used in the applications that themicrofluidic devices are designed for. For example, the sample volumethat is used is from sub-microliter up to 10 microliters for massspectrometer analysis using electrospray. As will be described ingreater detail hereinafter, the sample material is held in the reservoir60 and is then transported within the microfluidic channel 30 to thenozzle 50 where the sample materials are finally discharged through theopen second end 34. The outside diameter of the protruding nozzle 50also accordingly increases in a direction away from the tip portion 52thereof. By forming the reservoir 60 or input port at the first surface22 opposite to the second surface 24, where the nozzle 50 is formed, asample can easily be fed into the microfluidic channel 30 by injectingor otherwise disposing the sample into one or more reservoirs 50 andthen transporting the sample through the associated microfluidic channel30 using techniques described in greater detail hereinafter.

Turning now to FIG. 3, the microfluidic device 10 can be fabricated sothat it finds particular utility as a means for electrospray ionizationof analytes for mass spectrometer analysis. Electrospray is achieved bysubjecting the nozzle 50 to a voltage so that liquid and analytes (the“sample”) emerge to a high electric field. For this particularapplication, the microfluidic device 10 includes a conductive region 70formed on at least a portion of the nozzle 50 and optionally, theconductive region can extend onto the second surface 24. For example,the area around each nozzle 50 up to the extreme end of the nozzle 50 ismetallized by evaporation techniques, printing techniques, or othersuitable techniques known in the art to form the conductive region 70.Because the nozzle 50 in the illustrated embodiment has a conical shape,the conductive region 70 takes the form of a ring-shaped metal layerwith the nozzle 50 being in the center thereof. The thickness of theconductive region 70 can vary depending upon the precise application;however, the conductive region 70 should have a sufficient thickness sothat when an electric voltage is applied to the conductive region 70,the sample material (i.e., a liquid) within the microfluidic channelvaporizes and therefore can be used in electrospray or nanosprayapplications, such as electrospray ionization of analytes for a massspectrometer. The microfluidic device 10, in this example, provides alow cost, disposable electrospray interface capable of nanospray. Thisdevice can be fabricated to accommodate more than one sample input inorder to multiplex several separation instruments to a single massspectrometer.

Each of the conductive regions 70 formed around the nozzles 50 isconnected to one or more electrical contacts 80 formed at one edge ofthe substrate body 20. More specifically, the electrical contacts 80 arepreferably in the form of conductive pads (i.e., metallized tabs) thatare formed on the second surface 24 of the substrate body 20. FIG. 3shows one exemplary method of electrically connecting the conductiveregions 70 with the electrical contacts 80. In this exemplaryarrangement, one conductive region 70 is electrically connected via anelectrical pathway 90 to one electrical contact 80. The electricalpathway 90 simply provides an electrical pathway between the conductiveregion 70 and the electrical contact 80 and is therefore formed of aconductive material (e.g., a metal). For example, the electrical pathway90 can be in the form of a thin conductive film. By reducing the outsidediameter of the tip portion 52 of the nozzle 50 (e.g., to about 50 μm to80 μm), the voltage required to generate the spray is lowered. Accordingto one exemplary embodiment, the voltage used to form the spray is about5-6 KV for a tip portion 52 having an outside diameter from about 50 μmto 80 μm. It will be appreciated that larger sized outside diameters canbe used; however, this will require a greater voltage to be applied tothe nozzle 50 in order to form a spray.

It will be appreciated that more than one conductive region 70 can beelectrically attached to one electrical contact 80 using separateelectrical pathways 90 or using a network of electrical pathways or acomplete metal film. However, in this embodiment, when an electricvoltage is applied to the one electrical contact 80, the electricvoltage is applied to each of the conductive regions 70 that iselectrically connected to the one electrical contact 80. Thus, theelectric voltage can not be selectively delivered to individual nozzles50 in this particular embodiment.

Now referring to FIGS. 4-5, an exemplary microfluidic device 100according to a second embodiment is illustrated. The microfluidic device100 is similar in some respects to the microfluidic device 10 of FIGS.1-3. The microfluidic device 100 includes a substrate body 110 that isformed of a polymeric material and includes a first face 120 and asecond opposing face 130. Unlike the embodiment illustrated in FIGS.1-3, the first and second faces 120, 130 are not substantially planarsurfaces but rather are non-planar in nature due to each of the faces120, 130 having a number of recesses and protrusions formed therein.

The microfluidic device 100 has at least one microfluidic channel 140formed therein between the first face 120 and the second face 130 suchthat the microfluidic channel 140 extends completely through a thicknessof the substrate body 110 from the first face 120 to the second face130. The microfluidic channel 140 is thus open at both a first end 142at the first face 120 and at a second end 144 at the second face 130.The first face 120 includes a first perimeter wall 122 that extendsaround a perimeter of the microfluidic device 100 at the first face 120thereof. In the exemplary embodiment, the microfluidic device 100 isgenerally square shaped; however, this is merely one exemplary shape forthe microfluidic device 100 as the microfluidic device 100 can assumeany number of different shapes. Within the boundary of the firstperimeter wall 122, one or more reservoir walls 124 are formed with thenumber of reservoir walls 124 equal to the number of microfluidicchannels 140 formed in the substrate body 110. Each reservoir wall 124partially defines a reservoir 160 that is designed to hold a samplematerial and the reservoir wall 124 therefore also defines the first end142 of the microfluidic channel 140. Both the first perimeter wall 122and the one or more reservoir walls 124 extend above a generally planarsurface 126 (i.e., a floor) of the first face 120 in this embodiment. Asubstantial portion of reservoir 160, which is defined at the first end142 of the microfluidic device 140, is therefore formed above the planarsurface 126.

The second end 144 of the microfluidic channel 140 is formed in aprotrusion 170 that extends outwardly from the second face 130. As withthe prior embodiment, the protrusion 170 preferably has a tapered shape(inward taper) such that it forms a generally conical structure with theopen second end 144 being formed at an apex of the conical structure.The tapered protrusion 170 therefore acts as a nozzle that can dischargea sample that is loaded into the microfluidic channel 140 (e.g., in thereservoir 160). The nozzle 170 is therefore part of the microfluidicchannel structure since the microfluidic channel 140 is formedtherethrough and terminates at the nozzle opening.

The second face 130 is also not substantially planar but rather includesa second perimeter wall 132 that extends at least partially around aperimeter of the second face 130. The second face 130 does contain afloor 134 that is substantially planar. Between the second perimeterwall 132, one or more nozzle base sections 180 are formed with thenumber of nozzle base sections 180 being equal to the number ofmicrofluidic channels 140. The nozzle base sections 180 are integrallyformed with and extend outwardly from the floor 134 and in theillustrated embodiment, each nozzle base section 180 has a generallyannular shape. However, the shape of the nozzle base section 180 is notlimited to an annular shape and instead can have any number of shapes,including a conical shape or a tapered shape or any other regular orirregular shape. According to one embodiment, a plane that contains theupper edge of the second perimeter wall 132 generally cuts through theinterface between the nozzle base section 180 and the nozzle 170. Thenozzle 170 therefore extends beyond the upper edge of the secondperimeter wall 132. According to one embodiment, the diameter of thereservoir 160 is about equal to the outside diameter of the nozzle basesection 180; and therefore, an outside diameter of the reservoir wall124 is greater than the outside diameter of the nozzle base section 180.

The specific configurations of the nozzle 170 and the microfluidicchannel 140 are best shown in FIG. 5. As illustrated, the first end 142of the microfluidic channel 140 is in the form of the reservoir 160. Adistal end of the reservoir 160 has an inwardly tapered constructionthat leads to an intermediate channel section 146. A substantial lengthof the intermediate channel section 146 is formed in the nozzle basesection 180. The intermediate channel section 146 also has a taperedconstruction in that it tapers inwardly toward the nozzle 170 defined atthe second end 144 of the microfluidic channel 140. Thus, the dimensionsof the microfluidic channel 140 are greatest at the first end 142 andare at a minimum at a tip portion 172 of the nozzle 170. In oneembodiment, the microfluidic feature formed in the device 100 beginningwith the reservoir 160 and terminating with the nozzle 170 is generallycylindrical in shape along its length. According to one exemplaryembodiment, the open second end 144 of the microfluidic channel 140formed at the tip portion 172 has an inside diameter equal to or lessthan 100 μm, preferably equal to or less than 50 μm and more preferably,equal to or less than 20 μm; and an outside diameter of the nozzle, asmeasured at a tip portion thereof, is less than about 150 μm andpreferably is equal to or less than about 100 μm, and more preferablyequal to or less than 50 μm. The inside diameter of the microfluidicchannel 140 varies along its length due to its tapered construction. Forexample, the inside diameter of the microfluidic channel 140 opensgradually in a direction away from the nozzle 170 to about severalhundred μm as the microfluidic channel 140 traverses through thethickness of the substrate body 110 and eventually, the microfluidicchannel 140 is formed to a diameter of about 1.5 mm to define thereservoir 160. The length of the microfluidic channel 140 can betailored in view of the construction details of the microfluidic device100 and the potential applications of the device 100. In one example,the length of the microfluidic channel 140 is about 3 mm; however, thiswill vary depending upon the thickness of the device 100, the amount ofsample that is to be loaded into the device, etc.

As with the first embodiment, the microfluidic channel 140 is formed ina substantially perpendicular manner in the substrate body 110 since themicrofluidic channel 140 is formed substantially perpendicular to boththe first and second faces 120, 130. While, the nozzle 170 extendsbeyond a plane containing the distal edge of the second perimeter wall132, the distal end of the reservoir wall 124 preferably lies within thesame plane that contains the distal edge of the first perimeter wall122. This orientation permits a cover (e.g., thin polymeric cover sheet)or seal member to be disposed across the distal edge of the firstperimeter wall 122 and the distal ends of the reservoir wall 124 toeffectively seal the sample material within the reservoir 160, as willbe described hereinafter.

One will appreciate that one of the advantages of the device 100 is thatit is formed as a one piece construction in contrast to conventionaldevices which have multiple layers bonded together. In theseconventional devices, the microfluidic channel is closed by the bondingof one layer over another layer. In other words, two separate layers areneeded to define the complete channel. Because the present device 100 isinjection-molded, separate bonded layers are not required.

It will be understood that the present configurations that areillustrated herein with reference to FIGS. 1-5 are merely exemplary innature and are intended to merely convey exemplary embodiments. Variousmodifications can be performed to the microfluidic devices dependingupon a number of different considerations, including manufacturingconsiderations. For example, the nozzle structures do not necessarilyhave to have conical shapes; however, for ease of manufacturing, aconical shape or the like is generally preferred.

According to another aspect of the present application, variousmanufacturing methods are disclosed herein for manufacturing themicrofluidic array devices illustrated in FIGS. 1-5. In general terms,exemplary manufacturing processes disclosed herein permit microfluidicnozzle array devices to be manufactured having microscale nozzledimensions (e.g., a nozzle tip opening having a diameter equal to orless than 100 μm, preferably equal to or less than 50 μm and morepreferably, equal to or less than 20 μm; and an outside diameter of thenozzle, as measured at a tip portion thereof, is less than about 150 μmand preferably is equal to or less than about 100 μm, and morepreferably equal to or less than 50 μm) and also the presentmicrofluidic array devices are particularly suited to inexpensivefabrication methods. More specifically, the microfluidic array devicesof the present application can be manufactured by injection molding asuitable thermoplastic using conventional injection molding techniques.Suitable thermoplastics include polycyclic olefin polyethylenecopolymers, poly methyl methacrylate (PMMA), polycarbonate, polyalkanes,polyacrylate polybutanol co-polymers, polystyrenes, and polyionomers,such as Surlyn® and Bynel®. Polycyclic olefin polyethylene co-polymersare particularly suitable for use in an injection molding process.Various grades of such polymers are commercially available from Ticonaunder the trade name Topas® (which is a polyethylene-polycyclic olefinco-polymer). Furthermore, polybutyl terephthalate (PBT) can be used, aswell as polyamides, such as nylons of different grades (nylon 6-6,nylon, 6 nylon 6-12, etc.); polyoxymethylene (POM) and other acetylresins; and other resins with melt viscosity comparable to PBT and otherproperties similar to the other suitable polymers disclosed herein.Generally, polymers that are suitable for use in the present injectionmolding process include those thermoplastic polymers with a relativelylow melt viscosity and these polymers preferably also have a highchemical purity (preferably the polymers are without more than a fewpercent of particulate additives and are chemically inert). Othersuitable polymers include thermoplastics blended with a lubricant (e.g.,liquid crystalline polymers) added to help the flow and therefore thisadditive acts as a processing aid and other liquid crystalline polymerscontaining polymers such as Zenite® (DuPont Company) and the like can beused and polymers (both commercially available and non-commerciallyavailable) that have high chemical purity, high chemical resistivity andthermal stability are also suitable. In some applications,injection-moldable elastomers may also be suitable.

In order to manufacture the present microfluidic array devices usinginjection molding techniques, a mold or mold insert must first befabricated. The following description of the mold is merely exemplaryfor one type of mold construction which is oversimplified in terms ofits construction in order to illustrate certain details of overallmolding process. However, one of skill in the art will appreciate thatthe mold structure is readily changeable and is dictated by the desiredconstruction of the microfluidic device and more particularly, thedesired construction of the microfluidic channels based on the shape,dimensions and other properties thereof.

The mold typically is formed of several parts that mate with one anotherto form an assembled mold. The mold or mold insert is typically formedas a negative impression of whatever channel architecture or devicefeatures are desired in the microfluidic array device. A polymericmaterial is injected into the mold and then the polymeric material iscured to form the microfluidic array device which is then removed fromthe mold. Typically, the mold is formed of two mold dies that matetogether in a sealed manner and therefore after the microfluidic devicehas been formed and is sufficiently cooled, the two mold dies areseparated to permit access and removal of the microfluidic array device.

The mold (i.e., mold dies) or mold insert can be prepared from anynumber of materials that are suitable for such use, such as metal,silicon, quartz, sapphire and suitable polymeric materials; and formingthe negative impression of the channel architecture can be achieved bytechniques, such as photolithographic etching, stereolithographicetching, chemical etching, reactive ion etching, laser machining, rapidprototyping, inkjet printing and electroformation. Withelectroformation, the mold or mold insert is formed as a negativeimpression of the channel architecture by electroforming metal and themetal mold is polished (preferably to a mirror finish).

For non-metallic molds for injection molding, the mold can be made of aflat, hard material such as Si wafers, glass wafers, quartz or sapphire.The microfluidic design features can be formed in the mold throughphotolithography, chemical etching, reactive ion etching or lasermachining (which is commonly used in microfabrication facilities). Inaddition, some ceramics can be used to fabricate the mold or moldinsert.

Molds can also be fabricated from a “rapid prototyping” techniqueinvolving conventional ink-jet printing of the design or directlithography of resists, such as Su-8 or direct fabrication of the moldwith photopolymers using stereolithography, direct 3-dimensionalfabrication using polymers, and other similar or related techniques thatuse a variety of materials with polymers. A resulting polymer-based moldcan be electroformed to obtain a metallic negative replica of thepolymer-based mold. Metallic molds are particularly appropriate forinjection molding of polymers that require the mold itself to be heated.One commonly used metal for electroforming is nickel, although othermetals can also be used. The metallic electroformed mold is preferablypolished to a high degree of finish or “mirror” finish before use as themold for injection mold. This finish is comparable to the finishobtained with mechanical polishing of submicron to micron size abrasives(e.g., diamond particles). Electropolishing and other forms of polishingcan also be used to obtain the same degree of finish. Additionally, themetallic mold surface should preferably be as planar and as parallel asthe Si, glass, quartz, or sapphire wafers. In one exemplary embodiment,the metallic mold is polished to a highly polished finish by using 1micron diamond particles to provide a finish that is close to amirror-like finish.

The present applicant has discovered that injection molding techniquesusing a mold fabricated of hardened steel or other metals can be used tomanufacture polymeric microfluidic devices having an array of micronsized nozzle structures with a nozzle opening having a diameter equal toor less than 100 μm, preferably equal to or less than 50 μm and morepreferably, equal to or less than 20 μm; and an outside diameter of thenozzle, as measured at a tip portion thereof, is less than about 150 μmand preferably is equal to or less than about 100 μm, and morepreferably equal to or less than 50 μm. FIG. 6 is a perspective view ofa mold construction 200 that is constructed to injection mold amicrofluidic nozzle array device, as shown in FIG. 1, having theaforementioned dimensions and properties. Once again, the mold 200 isformed as a negative impression of the microfluidic device that is to beformed. The mold 200 includes a first mold die or part 210 and a secondmold die or part 230 that are constructed so that they are complementaryto one another and mate with one another to form an injection moldassembly that is used to form a microfluidic nozzle array device,similar to device 10 illustrated in FIG. 1. The mold 200 is preferablyformed by electric discharge machining (EDM).

The first mold die 210 has a first face 212 that includes asubstantially planar surface. The first face 212 has a recessed section214 formed therein. The recessed section 214 generally defines the outerperipheral shape of the microfluidic device and also the depth of therecessed section 214 defines the thickness of the microfluidic device(except in areas where the nozzles are formed). Because the microfluidicdevice typically has a square or rectangular shape, the shape of therecessed section 214 will be the same or similar. For example, theillustrated recessed section 214 is generally square shaped. The firstmold die 210 also includes a plurality of upstanding contoured pins 216that are spaced across a floor of the recessed section 214. The shape ofeach pin 216 directly corresponds to the shape of the microfluidicchannel that will be formed when the mold 200 is closed and thepolymeric material is injected. More specifically, a base section 217 ofthe pin 216 corresponds to the reservoir of the microfluidic channel; anintermediate section 218 corresponds to the intermediate section of themicrofluidic channel and a conical tip section 219 of the pin 216corresponds to the second end of the microfluidic channel that is formedin the tip portion of the nozzle. As a result, the dimensions of the pin216 are greatest at the base section 217 and the pin 216 tapers inwardlyto the conical tip section 219 thereof. The spacing of the pins 216directly correlates to the spacing of the microfluidic channel/nozzlestructure and therefore, the pins 216 are preferably spaced in arrays.

Now referring to FIGS. 6-7, the second mold die 230 has a first face 232that mates with the first face 212 of the first mold die 210. The firstface 232 is substantially planar with the exception that a plurality ofapertures 234 are formed in the second mold die 230. The apertures 234are arranged according to a predetermined pattern that corresponds tothe arrangement of the pins 216. The apertures 234 are sized so thatthey receive at least a portion of the conical tip sections 219 (about500 μm in length in one embodiment) of the pins 216 when the first andsecond mold dies 210, 230 mate with one another. The apertures 234 arethemselves contoured so that the apertures 234 taper inwardly with alower portion 235 of each aperture 234 having a conical shape so as toform the conical nozzle of the microfluidic device. When the first andsecond molds 210, 230 mate together and the pins 216 are received in theapertures 234 according to one embodiment, the tip sections 219 of thepins 216 extend completely to the bottom of the apertures 234 andcontact the body of the second die mold 210 that defines the closed endsof the apertures 234. The mold 200 of FIG. 6 is constructed to generallyproduce the microfluidic device 10 of FIG. 1.

FIG. 7 shows a cross-sectional view of a mold that is constructed toproduce the microfluidic device 100 of FIG. 4. For purposes of ease ofillustration and simplification, the reference numbers of FIG. 6 will becarried over to the description of FIGS. 7-9 since each of theseillustrated molds includes first and second mold dies. It will beunderstood that the features that are formed as part of the first andsecond mold dies 210, 230 dictate the dimensions and shape of thefeatures of the resulting microfluidic device.

It will therefore be appreciated that after the first and second molddies 210, 230 are closed and any preparation steps that are necessaryfor the injection molding process are taken, the first faces of thefirst mold die 210 and the second mold die 220 seat against one anotherto effectively seal the recessed section 214 and the polymeric material(typically a resin) is then injected into the closed space that isdefined in part by the recessed section 234. FIG. 7 shows across-sectional view of the first and second mold dies 210, 230 in aclosed position with the tip section 219 of one pin 216 received withinthe aperture 234 and more specifically into the conically shaped lowerportion 235 of the aperture 234. Because the first and second mold dies210, 230 are negative impressions of the resultant microfluidic device,the microfluidic channel will take the form of the pin 216 and thenozzle of the microfluidic device is formed by the conically shapedlower portion 235. More precisely, the nozzle is formed by resin fillingcompletely the space between the tip section 219 of pin 216 and the tipsection of the conically shaped lower portion 235. As previouslymentioned, in this embodiment, the tip section 219 of the pin 216 andthe tip of the second mold die 230 that is formed in the conically lowershaped portion 235 are in contact with one another.

Mold 200 is intended to be used a number of times over a period of timeto produce a great number of microfluidic devices and therefore thematerial that is selected for the fabrication of the mold 200 should bedone so accordingly. In other words, a material should be selected thatpermits microscale features to be formed in the microfluidic device andalso permits a great number of microfluidic devices to be formed usingthe mold 200. One material that is suitable for use in fabricating themold 200 is hardened steel. With conventional machining technologies,such as metal turning and electric discharge machining (EDM), thedimensions of the tip section 219 of the pin 216, which forms the nozzleopening, can be limited. For example, the dimensions (i.e., the diameterand length) of the tip section 219 can be limited due to manufacturingconsiderations. The available manufacturing techniques permit theoutside diameter of the nozzle to be formed to about 50 μm since it ispossible to inject mold a resin into the space between the tip section219 and the conically lower shaped portion 235. In some areas, thisspace is only on the order of about 15 μm due to the desired dimensionsof the nozzle and the microfluidic channel.

While, the first mold die 210 is illustrated as having a square shape,it will be appreciated that the first mold die 210 can be formed to haveany number of different shapes so long as the shapes of the first molddie 210 and the second mold die 230 permit these two components to matewith one another.

However, there are techniques available to injection mold a nozzleopening having smaller dimensions than the aforementioned dimensions.FIG. 8 illustrates one possible injection molding arrangement toaccomplish this task and produce nozzles having nozzle openings that areeven smaller than the tip section 219 of the pin 216. In FIG. 8, thereis a gap 240 between the tip section 219 and the conically shaped lowerportion 235 after the first and second mold dies 210, 230 have beenassembled. When the polymeric material (e.g., a resin) is injected (in amolten state) into the conically shaped lower portion 235, the pressureof the injected resin is adjusted such that the resin does not fill theentire space in the gap 240 and an opening (space) remains at the tip ofthe resulting molded nozzle since sufficient pressure is not present todisplace the resin to the lowermost section of portion 235. Using thistechnique, the diameter of the tip section 219 of the pin 216 can begreater than 20 μm since the opening of the nozzle and the outsidediameter of the nozzle are no longer defined by the dimensions of thecorresponding parts of mold but rather are defined by a combination ofmold dimensions, gap dimension and injection pressure. In this manner,the pins 216 do not have to be manufactured to have a tip section 219 onthe order of 20 μm in order to form a nozzle opening of the samedimension. Instead, the tip section 219 can have a diameter greater thanthe diameter of the nozzle opening that is ultimately formed in thenozzle as a result of the injection molding process.

FIG. 9 illustrates one exemplary method of overshooting the injectedresin into the gap 240 formed between the tip section 219 and the tip ofthe conically shaped lower portion 235. The nozzle opening 215 isdefined by pressure used to inject the molten resin and the dimensionsof the gap 240. By controlling these parameters, the dimensions of thenozzle opening can be controlled.

Injection molding as a manufacturing technology for polymer parts islow-cost at high-volume production. However, there is considerable costinvolved in the production of the mold itself, especially for amicrofluidic nozzle design which has micron sized features and thereforeis a demanding design in terms of producing a mold. If the microfluidicnozzle array device is arranged to have the same pattern as themicrotiter plate so that commercial robotic liquid dispensing equipmentcan be used to fill the reservoirs of the microfluidic channels withsamples, then tiling or combining a number of smaller microfluidicnozzle array devices (i.e., subunits) to form a larger structure can beused since the microtiter plates consist of regularly spaced sampleinput points in a grid pattern. For example, the microfluidic nozzlearray devices can be formed and then combined with one another toproduce a structure that has the desired number of sample reservoirs(also referred to as sample wells or sample inputs) to receive a desirednumber of samples. For example, some common microfluidic devices contain96 sample reservoirs (8×12 grid); 384 sample reservoirs (16×24 grid);and 1536 sample reservoirs (32×48 grid). The tiling can be done bynumber of known conventional means, including by permanently bondingadjacent tiles together by melt bonding, welding, gluing, etc. In otherwords, any suitable method or technique for joining polymer structurestogether can be used. The subunit structures can be formed as individualsubunit tiles (see FIGS. 17-18) or the subunit structure can be in theform of an elongated strip that includes a number of rows of nozzles.For example, the strip can be formed to include 2 rows of spaced apartnozzles.

Alternatively, the user can be supplied with a base plate that has anumber of features formed therein to permit nozzle subunit structures tobe inserted into and retained by the base plate. For example, the baseplate can contain pre-defined receptacles that receive the nozzlesubunit structures in such a way that the nozzle subunit structures aresecurely held within the base plate and are arranged according to adesired pattern. One or both of the base plate and the nozzle subunitstructures can contain interlocking features to provide an interlockingconnection between the base plate and the nozzle subunit structures. Inthis embodiment, the base plate functions as a base on which the finalmicrofluidic nozzle array device can be constructed by arranging anumber of nozzle subunit structures together and then securely holdingthese subunit structures within the base plate. One exemplary structurefor releasably holding the nozzle subunits in an interlocked manner isillustrated in FIG. 17 and is discussed in greater detail hereinafter inthe discussion of Example 3.

There are a number of advantages that are obtained by tiling orotherwise combining a number of nozzle subunit structures into amicrofluidic nozzle array device of greater dimension. First, the costof manufacturing the mold for the smaller nozzle subunit structure issubstantially less than the cost of manufacturing a mold for the entiregrid of the microfluidic nozzle. Also, the cost of mold replacement isalso substantially reduced in the case that only one pin in the mold isdamaged. Second, the utility of the nozzle array is made more flexible.If an experiment does not require all of the reservoirs (e.g., 96) ofthe microfluidic device to be filled, only the needed number of nozzlesor a number close thereto can be inserted into the base plate. At thesame time, this construction still permits robotic dispensing ofsamples. For example and according to one exemplary embodiment, onenozzle subunit structure contains 4 reservoirs and therefore, if theexperiment only requires 60 reservoirs, then only 15 nozzle subunitstructures are inserted into the base plate. In this manner, thepotential waste or inefficiency related to each microfluidic device iseliminated or greatly reduced because the number of unused reservoirs isgreatly reduced or entirely eliminated.

Third, when the microfluidic nozzle array device is used forelectrospray or nanospray in front of a mass spectrometer inlet, acommon configuration is to have the nozzle spray “off-axis”, i.e., thenozzle sprays in a direction perpendicular to the inlet. Since thenozzle has to be placed in close proximity to the inlet (e.g., typicallywithin an inch), there is often times not enough room in front of theinlet to accommodate the entire microtiter plate. FIG. 10 illustrateshow a tiled microfluidic nozzle array microtiter plate can be used forelectrospray in the off-axis configuration. A tiled microfluidic nozzlearray 300 arranged in a 96 well microtiter plate format is broken upinto strips 302 with two rows of 12 nozzles 310 each. One of the strips302 is broken away or is otherwise removed from the others and istransferred (as indicated by arrow 320) to a nozzle mount (not shown) infront of a mass spectrometer inlet 330 of a mass spectrometer 340. Thenozzle mount holds the strip 302 and has at least an x-y translationstage such that each of the nozzles can be placed in an optimal positionwith respect to the mass spectrometer inlet 330 for spraying of thesample material that is contained within the microfluidic channelassociated with the selected nozzle. The direction of the spray isperpendicular to the mass spectrometer inlet 330. In schematic drawingof FIG. 10, the nozzles 310 are positioned below the centerline of themass spectrometer inlet 330 and the spray is in the direction out of thesurface of the drawing figure. It will be appreciated that the strips302 that are still in tact can be used in future applications either byusing the entire structure of joined strips 302 or by detaching one ormore strips 302 for use in a given application depending upon theprecise application and what the requirements for the application are interms of the number of nozzles 310 that is needed.

The microfluidic nozzle array devices disclosed herein are suitable foruse in a number of different types of applications.

For purposes of illustration only, some of the exemplary applicationswill be disclosed with reference to the microfluidic nozzle array device100 illustrated in FIGS. 4-5; however, it will be understood that any ofthe devices disclosed herein can be used in place of device 100.

The microfluidic nozzle array device 100 is particularly suited for usein nanospray/electrospray applications. Electrospray is the techniquethat enables a liquid sample to be vaporized and ionized for massspectrometry analysis. The electrospray process takes place in ambientpressure. Conventional electrospray utilizes a capillary with arelatively large inside diameter (i.e., about 50 μm) to deliver theliquid sample to the entrance of the mass spectrometer. The liquid thatis flowing out of the capillary is vaporized under the influence of anelectric field generated by placing a high voltage (e.g., 4-5 KV) on ametallic conductor close to the capillary opening and a ground planeopposite the capillary opening, or vice versa. Dry nitrogen flowsthrough concentric tubing to the capillary to help nebulize the liquidflowing out of the capillary. The flow of the liquid inside thecapillary is driven generally by a pump, such as a syringe pump.

For the nozzle array of the present microfluidic device 100 to be usedas individual nanospray sources, the reservoir 160 on the opposite sideof the nozzle opening is filled with a sample to be sprayed. Before thespray, the reservoir has to be sealed so that the reservoir is liquidtight. In other words, the open end of reservoir 160 (i.e., the openfirst end 142 of the microfluidic channel 140) must be sealed. Thesealing of the open end of the reservoir 160 can be accomplished in anumber of different ways that each provides a satisfactory liquid tightseal of the reservoir and permits the sample to be transported withinthe channel 140. FIGS. 11-13, illustrate a number of exemplary ways toprovide the desired liquid tight seal of the reservoir.

For example, FIG. 11 illustrates a first sealing technique in which theopening of the reservoir 160 (i.e., the first end 142 of themicrofluidic channel 140) is sealed with an elastic cover sheet 400. Theelastic cover sheet 400 is preferably in the form of an elasticpolymeric cover sheet. In the microfluidic nozzle array device 100, thepolymeric cover sheet 400 is coupled to the reservoir wall 124 so thatthe polymeric cover sheet 400 extends completely across the open end ofthe reservoir 160. A mechanical plunger 410 or the like can be used toapply a force to the polymeric cover sheet 400 to force the sample alongthe length of the microfluidic channel 140 and ultimately out of thenozzle opening (second end 144 of the microfluidic channel 140) in acontinuous stream, generally indicated at 430. The discharged continuousliquid stream of the sample is then vaporized under the influence of anelectric field. The general direction of movement of the polymeric coversheet 400 and the plunger 410 is illustrate by arrow 420.

Another sealing technique is illustrated in FIG. 12. According to thistechnique, a movable sealing member 400 is provided and is formed of asealing base 422 for sealing the opening of the reservoir and a rod orplunger 444 that is attached to the sealing base 442. The dimensions ofthe sealing base 442 are greater than the dimensions of the open end ofthe reservoir 160 and therefore, the sealing base 442 seats against thereservoir wall 124 and completely extends across the open end of thereservoir 160. The sealing base 442 is formed of a suitable elasticmaterial to permit the sealing base to locally deform when a force isapplied thereto. This elasticity permits the sealing base 442 to act asa temporary diaphragm that seals the reservoir as the sealing base 442is directed into the reservoir 160 itself.

When the sealing base 442 is pushed downward in the direction toward thenozzle 170, the sealing base 442 deforms as it is forced into the firstend of the microfluidic channel 140 (which is also the entrance to thereservoir 160). In the illustrated embodiment, the sealing base 442includes a flange 446 that has a greater diameter than the diameter ofthe other portions of the sealing base and therefore, when the sealingbase is inserted into the reservoir, the flange 446 intimately contactsthe inner surface of the reservoir wall 124 and forms the liquid tightseal between the sealing base and the reservoir. As the sealing base 442is inserted into the reservoir 160 and travels therein toward thenozzle, the sealing base 442 effectively forces the sample toward thesecond end 144 of the microfluidic channel 140, causing the sample to bedischarged through the nozzle opening defined thereat. There may be anair gap between the sample (e.g., a liquid) in the reservoir 160 and thesealing base 442 or a vent (not shown) can be incorporated into thesealing base 442 for air to be pushed out of the reservoir 160 when thesample is forced through the microfluidic device by the sealing base442. The vent can be fabricated using conventional vent technology inthat the vent should permit air passage, while being impermeable to theflow of liquid so that the sample is prevented from flowing through thevent and out of the reservoir 160.

It will be appreciated that the plunger 444 can either be manuallyoperated or it can be part of an automated system including an actuatoror the like which controls the movement of the plunger 444. All of theplungers 444 can be linked to a common actuator or the link so that uponactivation, the plungers 444 are all driven at the same time, resultingin the samples being concurrently transported through respectivechannels to respective nozzles.

Yet another sealing technique is illustrated with reference to FIG. 13in which a fluid carrying member 450 is provided. The member 450 has ahollow portion and is generally shaped to be complementary to the shapeof the reservoir 160 to permit the member 450 to seat against the upperedge of the reservoir wall 124. The member 450 includes a distal end 452which initially is positioned proximate to the open end of the reservoir160. At the distal end 452, a gasket 460 is provided and in theillustrated embodiment, the gasket 460 is in the form of a sealingO-ring or the like. The gasket 460 serves to provide a seal between thedistal end 452 and the reservoir wall 124 to prevent from escapingbetween this interface when the sample is transported in the followingmanner. Because the member 450 is at least partially hollow, the gasket460 is disposed around the bore that extends through the member 450.

In this embodiment, the sample is moved within the microfluidic channel140 by conducting a fluid through the member 450 (more specifically, thebore thereof) to effectively force the sample through the microfluidicchannel 140 to the nozzle 170 where the sample is discharged incontinuous stream 430. The fluid is preferably a high-pressure gas, suchas air or dry nitrogen gas that is delivered from a source that is influid communication with the piston bore. The flow direction of thefluid is generally indicated at 470. In another embodiment, the fluidcan be the liquid sample fed into the microfluidic channel 140 tocontinuously push liquid out through the nozzle.

It will be appreciated that a protective cover (not shown) can be placedat the distal end 452 of the fluid carrying member 450 to prevent samplefrom contacting the inner surfaces of the piston bore. The protectivecover must be permeable to the fluid that flows through the bore andinto the reservoir 160 to transport the sample along the microfluidicchannel 140. For example, the protective cover can be in the formed of athin polymeric film that is gas permeable, while at the same time beingimpermeable to liquid flow. In this manner, the sample can not contactthe bore itself. The use of such a protective cover is not requiredsince the injected fluid that flows through the member 450 can push theliquid sample out by applying a force to the air gap between the sampleand the surrounding structure.

A more conventional fluid delivery mechanism can be used with the device100. In this embodiment, a stopper is inserted into the reservoir 160,with the stopper having a bore formed therethrough which is incommunication with the reservoir 160. A capillary is inserted throughthe bore and the liquid sample is injected into the reservoir throughthe capillary from a source external to the capillary. In thisembodiment, the sample is not stored in the reservoir 160 but rather isdelivered to the channel 140 by being injected into the reservoir 160through the capillary.

As previously mentioned, the front face of the nozzle array is madeelectrically conducting by a thin film of metal or conducting polymer.When an electric field of appropriate strength is applied to the nozzle(e.g., as by the arrangement illustrated in FIG. 3), the liquid and theanalytes it carries (i.e., the sample) are vaporized as they aredischarged through the nozzle opening. Liquids that are suitable for usein electrospray mass spectrometry analysis include but are not limitedto acetonitrile, methanol, ammonium acetate, and other volatile liquids.Since the inside diameter of the nozzle is less than about 20 μm, theamount of material flowing out of the nozzle to be vaporized is lessthan the amount that is typically used in a conventional electrosprayoperation. Also, the outside of 50 μm creates a strong enough electricfield for vaporization with applied voltage below about 6 KV.

The use of a nebulizing gas to assist in the vaporization process istherefore not needed; however, if nebulizing gas is needed, channelsconducting dry nitrogen gas to the nozzle opening may be easily added ina polymer substrate attached to the front of the nozzle array. FIGS.14-15 are a top plan view and a cross-sectional view, respectively, of amicrofluidic nozzle array device 500 in combination with a substrate 510having gas conduits 520 formed therein for nebulization. Themicrofluidic nozzle array device 500 can be similar to or identical toany of the exemplary microfluidic array devices disclosed hereinbefore.A gas outlet 522 is formed such that it is concentric with one nozzle530. The substrate 510 with the nebulizing gas channels can befabricated by an injection molding process during the injection moldingprocess that is used to the nozzle array device 500 itself or it can befabricated first and then later attached to (e.g., bonded) the nozzlearray device 500 as a separate component. The substrate 510 can beattached in any number of different ways including but not limited tousing an adhesive or meltingly bonding the two members along a boundaryzone.

In some instances, it may not be necessary to have the nozzle arrayconform to the microtiter plate sample well format. For example, thesample can be fed to the nozzle by the elutant of a high performanceliquid phase gas chromatography (HPLC) column. Since the reservoir sizein the nozzle array can be formed to arbitrary sizes, it can be formedso that the open end of the reservoir can receive one end of the HPLCcolumn or any plumbing for splitting the HPLC elutant for massspectrometry analysis. The reservoir side of the nozzle array can alsoconsist of injection molded features for splitting elutant for massspectrometry analysis. The driving force for the liquid sample analytesto flow through the nozzle opening in this case is the pressure-drivenliquid flow of the HPLC. Neither a pressure diaphragm nor an externalpressure-inducing mechanism is needed.

The microfluidic nozzle array devices disclosed herein are alsoparticularly adapted to be used as a nozzle array for opticalspectrometry. Since each microfluidic channel in the nozzle array deviceterminates with a nozzle opening having an inside diameter of 20 μm orless and the substrate of the nozzle array device is formed of apolymeric material which is generally hydrophobic, liquid inside themicrofluidic channel does not drip or be discharged out of the nozzlewithout external force being applied thereto. When light, eitherultraviolet or visible, is incident on the reservoir side of the array,the light will come out of the nozzle opening carrying the opticalspectroscopic information of the analytes contained within the liquid inthe microfluidic channel. The microfluidic channel and the nozzleopening thus provide an optical detection system without the use ofoptical windows. This is a significant advantage since the microfluidicnozzle array device does not have to be fabricated to incorporateoptical windows made of an optical material in its design. This resultsin reduced structural complexity for the microfluidic nozzle arraydevice and also a reduction in both cost and complexity relative to thefabrication of the microfluidic nozzle array device.

A 96 microtiter nozzle plate filled with samples can be placed in anultraviolet reader for a 96 microtiter plate and spectrophotometricinformation for each sample can be obtained with the reader. Aconventional microtiter plate used for UV spectrophotometry must have asample well bottom made of a special UV transparent material in order tohold the sample inside the well and transmit UV light at the same timeor a microtiter plate made of quartz must be used. The use of amicrotiter nozzle plate array plate according to one exemplaryembodiment thus allows two detection techniques for the samples in theplate without having to transfer the samples to other additional plates.

FIG. 16 is a cross-sectional view illustrating how the microfluidicnozzle array device 100 can be used for UV spectrophotometry. FIG. 16illustrates the microfluidic nozzle array device 160 in partial sectionshowing two nozzle structures for purposes of illustrating the use ofthe microfluidic nozzle array device 100 in UV spectrophotometry. Inthis exemplary arrangement, UV light is emitted from a source 540 andtravels toward the microfluidic nozzle array device 100 and is incidenton the reservoir side 120. The UV light travels through the reservoir160 and continues to travel along the length of the microfluidic channel140, both of which hold the sample (e.g., liquid and analytes). The UVlight travels through the nozzle opening 144 to a detector 550 that isdisposed such that it faces the side of the microfluidic nozzle arraydevice that contains the nozzles 170. The UV light carries thespectrophotometric information of the analyte is detected by thedetector 550 of the UV reader. In this manner, the formation ofperpendicular orientated microfluidic channels provides advantageouslypermits UV spectrophotometry to be carried out in an easy and convenientmanner since the microfluidic nozzle array device 100 can easily bedisposed between a UV light source and the detector 550 of the UVreader. Likewise, transmission fluorescence spectroscopy can be carriedout using the microfluidic nozzle array device 100.

Unlike conventional microfluidic devices where optical windows formed ofan optical material were fabricated in the devices, the substrate bodyof the present microfluidic nozzle array device does not have to beformed of an optically transparent material. This reduces the complexityof the fabrication process since this requirement is not present in themicrofluidic nozzle array device.

The present microfluidic nozzle array devices disclosed herein also canbe used in a wide range of other applications in which similarconventional devices have typically been used. For example, themicrofluidic nozzle array device can be used for spotting DNA or proteinarray on a substrate instead of using the conventional capillary wickingmethods that are now used with metallic capillaries. Presently, the DNAarray spotting is primarily carried out by “wicking” DNA fragments intoan open split end of a metallic capillary. To spot in an array format ona glass slide, the split end of the capillary is pressed slightly ontothe glass slide by a robotic arm or the like to facilitate thedeposition of the DNA fragments. On being lifted from the glass slide,the metallic capillary has a tendency to “spring” off the glass slide.As a result of this phenomena and other factors, it is common that about20% of spots in the array are deficient in some way, e.g., either thespot is bare or an inadequate amount of material has been deposited.Spotting is typically carried out with a row of eight to twelvecapillaries using an expensive machine and the capillaries are rinsedand reused for different DNA samples.

The present microfluidic nozzle array devices disclosed herein havesmaller nozzles openings (e.g., 20 μm or less) than conventional nozzleconstructions and a number of advantages can be realized using thepresent microfluidic nozzle array devices in comparison to theconventional metal capillaries. First, the injection-molded microfluidicnozzle array devices can be disposed of after each deposition. Thus, thetime consuming rinsing process is eliminated and there is no risk ofcross-contamination since the devices are not reused. Second, DNA orprotein molecules are not adsorbed on the walls of the polymeric nozzleas they are adsorbed on metallic surfaces. The spotting is thereforemore complete when the molecules leave the polymeric nozzle to bedeposited on the glass slide. Third, a two dimensional nozzle spottercan be manufactured inexpensively thereby greatly increasing the speedof the spotting operation. Fourth, the deposition of the DNA or proteinmolecules from the polymeric nozzle can be assisted by pumping themolecules out of the nozzle with high pressure air using one of theaforementioned devices and/or with an electric field for electrospray.

The microfluidic nozzle array device can also be used for spotting theplate for matrix-assisted laser desorption ionization (MALDI), replacingthe pipette and capillary spotting methods. For matrix-assisted laserdesorption and ionization mass spectrometry, a dominant analyticaltechnique for protein molecules and fragments of high molecular weight,the molecules to be analyzed are deposited on a layer of matrixmaterial, usually UV-absorbant molecules that can be vaporized by a UVlaser. The molecules of interest are thus carried into the gas phase andare ionized alongside the matrix molecules. Traditionally, the metallic(usually aluminum) MALDI plate is spotted manually with the use ofmicropipettes and more recently with capillaries. The efficiency of theionization process will be enhanced if the metallized polymeric nozzlesare used for spotting. The matrix material is first electrosprayed ontothe aluminum MALDI plate which is held at ground potential, whereas themetal coated nozzle is held at high voltage or vice versa. The moleculesof interest are then electrosprayed in a new nozzle onto the matrixmaterial. The spraying allows the matrix molecules and the molecules ofinterest to be more evenly intermingled with one another, thus enhancingthe efficiency of laser assisted desorption and ionization. The spottingof the MALDI plate may also be carried out with a two-dimensional arrayof nozzles for high throughput. Thus, the density of the nozzle arraycan be greatly increased and this permits the density of the spottingarray to be increased. Accordingly, more testing or experimental sitesare provided on the substrate as a result on the increased density inthe spotting. It will also be appreciated that an electric field canalso be used to assist in the spotting process. The electric field canbe generated by using the arrangement illustrated in FIG. 3 or by someother type of suitable arrangement.

One will further appreciate that the manufacturing methods disclosedherein that are based on injection molding techniques can be used tomake pipette tips for nano to picoliter dispensing. In other words, amold can be fabricated and resin can be injected into the mold to formpipette tips that have an elongated body and terminate in a tip sectionthat has a tip opening having an inside diameter of less than about 20μm (with the tip section having an outside diameter of less than about50 μm.

The following examples serve merely to illustrate several embodiments ofthe present microfluidic array devices and do not limit the scope of thepresent invention in any way.

Example 1

A polymeric microfluidic nozzle array device is fabricated using thetechnology disclosed herein is by first providing a mold designed for aninjection mold process. The mold is formed of a metal and a conicalsurface of the mold that defines the nozzle portion of the microfluidicdevice is polished with a diamond paste to form a highly polishedsurface. More specifically, the conical surface is polished with 1micron diamond particles to provide a close to mirror finish for thenozzle that is formed as part of the microfluidic device. Themicrofluidic device is fabricated by injecting polybutyl terephthalate(PBT) into the closed mold and then curing the formed structure and thenultimately removing the molded microfluidic nozzle array device from themold. The microfluidic nozzle array device is formed to have nozzlesthat have an average outside diameter of about 60 microns and an averageinside diameter of the tip (i.e., the diameter of the nozzle opening)being less than about 20 microns.

By polishing the conical surface of the mold that defines the nozzle,the outer surface of the nozzle is made much smoother and further theshape of the nozzles is more consistent from nozzle to nozzle and frommold run to mold run. By providing a smooth highly polished surface inthe conical portion, the friction of the resin flow is reduced and thisresults in an increase in the accuracy and efficiency of the injectionprocess. These techniques provide advantages when forming structureshaving very small dimensions, such as the nozzles of the presentmicrofluidic device which have microscale features.

The microfluidic nozzle array device is then used as an electrospraydevice for spraying a liquid sample that is disposed within themicrofluidic features formed in the microfluidic nozzle array device. Asdescribed in detail hereinbefore, the nozzle serves to spray the liquidsample into a fine mist through electric-field induced evaporation. Inthis example, a voltage of between 5-6 KV is applied to a conductiveregion formed around the nozzle tip in order to provide the necessaryelectric-field. The vaporized, ionized sample is then injected into aninlet of a mass spectrometer for analysis.

Example 2

A polymeric microfluidic nozzle array device is fabricated using thetechnology disclosed herein by first providing a mold designed for aninjection mold process. The mold is formed of a metal and a conicalsurface of the mold that defines the nozzle portion of the microfluidicdevice is polished with a diamond paste to form a highly polishedsurface. More specifically, the conical surface is polished with 1micron diamond particles to provide a close to mirror finish for thenozzle that is formed as part of the microfluidic device. Themicrofluidic device is fabricated by injecting polybutyl terephthalate(PBT) into the closed mold and then curing the formed structure and thenultimately removing the molded microfluidic nozzle array device from themold. The microfluidic nozzle array device is formed to have nozzlesthat have an average outside diameter of about 60 microns and an averageinside diameter of the tips (i.e., the diameter of the nozzle opening)being less than about 20 microns. The mold is constructed so that amicrofluidic nozzle array strip is formed having two rows of twelvenozzles each.

Upon removing the molded microfluidic nozzle array strip, the aboveprocess is repeated to form one or more other microfluidic nozzle arraystrips. The microfluidic nozzle array strips are then placed side byside and adjacent strips are detachably secured to one another byapplying an adhesive (e.g., glue) to an edge of the each of the strips.More specifically, the edges are heated so that the polymeric materialsoftens and then the adjacent strips are joined together along theseedges so that a fused bond results between the two edges that arebrought into contact. Preferably, the fused bond between adjacent stripsincludes a weakened section (e.g., a score line or the like can beformed along the bond or the thickness of the bonded interface sectionbetween the two strips can be of reduced thickness) so that one stripcan easily be detached from the other strip. Any remaining microfluidicstrips are attached in the same manner to form a single, tiledmicrofluidic nozzle array device that contains a weakened sectionbetween the adjacent bonded microfluidic devices. The number of bondedmicrofluidic nozzle array strips will vary depending upon the desiredoverall size of the microfluidic nozzle array device and moreparticularly, the desired overall number of reservoirs and nozzles pereach microfluidic device. In use, the single, tiled microfluidic nozzlearray device is broken apart into two or more sections which can be usedor can further be broken apart into additional smaller microfluidicdevices.

Example 3

A polymeric microfluidic nozzle array device is fabricated using thetechnology disclosed herein by first providing a mold designed for aninjection mold process. The mold is formed of a metal and a conicalsurface of the mold that defines the nozzle portion of the microfluidicdevice is polished with a diamond paste to form a highly polishedsurface. More specifically, the conical surface is polished with 1micron diamond particles to provide a close to mirror finish for thenozzle that is formed as part of the microfluidic device. Themicrofluidic device is fabricated by injecting polybutyl terephthalate(PBT) into the closed mold and then curing the formed structure and thenultimately removing the molded microfluidic nozzle array device from themold. The microfluidic nozzle array device is formed to have nozzlesthat have an average outside diameter of about 60 microns and an averageinside diameter of the tips (i.e., the diameter of the nozzle opening)being less than about 20 microns. The mold is constructed so that amicrofluidic nozzle array strip is formed having two rows of twelvenozzles each.

Upon removing the molded microfluidic nozzle array strip, the aboveprocess is repeated to form one or more other microfluidic nozzle arraystrips. FIG. 17 generally illustrates the concept of tiling or otherwisecombining a number of nozzle subunit structures into a microfluidicnozzle array device of greater dimension. A base plate 600 is providedand serves as the means for receiving a number of nozzle subunitsstructures, generally indicated at 610, in a manner in which the nozzlesubunit structures 610 are releasably interlocked with the base plate600. More specifically, the base plate 600 is a frame-like member havinga predetermined number of retaining rails 620 that are affixed at theirends to a pair of end walls 630. The rails 620 are spaced apart from oneanother so that open slots 640 are formed between adjacent rails 620.

As illustrated in FIGS. 17 and 18, each rail 620 has a number ofclamping features 650 formed as a part thereof and spaced along thelength of the rail 620. The clamping feature 650 includes side walls 652that are spaced apart from one another to define a retaining slot 660therebetween. The side walls 652 are disposed parallel to one anotherand extend upwardly from a floor 654 of the clamping feature 650. Thedistance between inner surfaces of the side walls 652 is selected so asto provide a frictional fit between a side wall of the nozzle subunitstructure 610 so as to secure the structure 610 to the base 600 while atthe same time permitting the structure 610 to be disengaged and easilyremoved from the base 600. Accordingly, the distance between the innersurfaces of the side walls 652 is equal to or slightly greater than awidth of the side wall of the structure 610 that is received within theretaining slot 660 between the side walls 652.

Alternatively, the entire length of the rail 620 can have a “U-shaped”cross-section with a retaining slot 660 being formed between two sidewalls 652 that are spaced apart from one another. In this embodiment,the entire rail 620 serves as locking member instead of discreteclamping features 650 that are spaced along its length.

In the illustrated embodiment, each nozzle subunit structure 610includes four nozzles 612 and four reservoirs (not shown) on theopposite side of the structure 610. For purpose of illustration only,the nozzles 612 are illustrated as facing away from the clampingfeatures 650 (such that the nozzles 612 are in a plane above theclamping features 650); however, the structure 610 can be releasablyinterlocked with the base 600 such that the nozzles 612 face in theopposite direction. In other words, the reservoirs at the opposite endof the microfluidic channel face away from the clamping features 650 andare located in a plane above the clamping features 650.

The nozzle subunit structures 610 are releasably interlocked with thebase 600 by inserting the two opposing side walls 611 of one nozzlesubunit structure 610 into retaining slots 660 of two adjacent rails 620that face another with an open slot 640 therebetween. One side wall 611can be inserted first and then the other side wall 611 can be insertedinto the other retaining slot 660 or both side walls 611 can be alignedwith the slots 660 and then the nozzle subunit structure can be presseddownward to effectively dispose the side walls 611 within the retainingslots 660. Because both the nozzle subunit structure 610 and the base600 are preferably formed of plastic materials and the dimensions of thestructures are carefully selected, a frictional fit results when theside walls 611 are received within the retaining slots 660. When theside walls 611 are received within the retaining slots 660, the nozzles612 and the reservoirs are received within the open slot 640 such thatthese elements are not obstructed by the base 600. In other words, thereservoir openings are clear so that samples can be injected orotherwise disposed within the reservoirs and also the nozzle openingsare clear so that the sample can be discharged.

In one embodiment, the base 600 is formed of a polymeric material and ismanufactured using an injection molding process such that the base 600is formed as a unitary structure. While a frictional fit is one mannerof releasably interlocking the nozzle subunit structures 610 to the base600, a small amount of adhesive may be used at the interface between theside walls 611 and the clamping features 650 to ensure that the nozzlesubunit structures 610 remain in place during various applications (whenthe base 600 may need to be turned upside down, etc.). Further, someapplications require that a force be applied to the backside of thenozzle subunit structure 610 (e.g., due to actuation of a plunger in thereservoir, etc.) and therefore it is desirable for the nozzle subunitstructures 610 to remain in place and not become dislodged from the base600 when this force is applied. Any number of suitable adhesives can beused and it will be appreciated that one type of adhesive is areleasable adhesive that permits the nozzle subunit structure 610 to beremoved from the base 600.

FIG. 19 illustrates another embodiment of base 600 that is very similarto the configuration illustrates in FIGS. 17-18. In this embodiment, theclamping features 650 are configured to receive two side walls 611 ofadjacent nozzle subunit structures 610. Thus, the distance between theinner surfaces of the side walls 652 is selected so that the width oftwo side walls 611 placed in intimate adjacent contact with one anotheris about equal to or slightly less than the distance between the innersurfaces of the side walls 652. In other words, the slot 660 isconfigured to receive and retain two side walls 611 of adjacent nozzlesubunit structures 610. To removeably couple the nozzle subunitstructures 610 to the base 600 according to this embodiment, one sidewall 611 is disposed within the slot 660 and then another side wall 611of an adjacent nozzle subunit structure 610 is disposed in the slot 660next to the other side wall 611, thereby providing a frictional fit thatresults in both adjacent nozzle subunit structures 610 being heldsecurely in place. Unlike the embodiment of FIGS. 17-18, this embodimentrequires that the two side walls 611 be disposed within one slot 660 toeffectively couple each nozzle subunit structure to the base 600.

It will be appreciated that other clamping members can be used besidesthe above described ones. For example, each clamping member can consistof a spring biased clip that receives side wall 611 in a frictionalmanner so as to retain and hold the side wall 611 in a releasablemanner. The clip can consist of two opposing plates that are hingedlyconnected at one end so as to bias the plates toward one another. Theside wall 611 is received at the opposite ends of the plates insertingthe side wall 611 between the plates and then directing the side wall611 between the plates toward the hinged end. The biasing action betweenthe plates ensures that the side wall 611 is securely gripped betweenthe plates, while at the same time can be removed by simply overcomingthe biasing force and lifting the side wall 611 upward until it is freeof the plates.

Referring now to FIGS. 20-21 in which another aspect of the presentinvention is shown. For purpose of illustration and simplification, onlythe nozzle 50 of the microfluidic device, such as device 10 of FIG. 1,is illustrated. The microfluidic channel 30 is generally shown and itwill be appreciated that the channel 30 includes at the opposite end areservoir (reservoir 60 of FIG. 1) where the sample is injected firstbefore passing into the nozzle 50. In this embodiment, the conicallyshaped nozzle 50 is injection molded, along with the other portions ofthe device 10, to be sealed at the apex end of the channel 30. In otherwords, a thin membrane 31 of material seals the second end 34, therebysealing the top portion 52 of the nozzle 50. In this example, the thinmembrane 31 is formed of the same injection moldable material that isused to form the rest of the microfluidic device 10. The thickness ofthe thin membrane 31 that seals the second end 34 of the channel 30 maybe from about a few μm to about 100 μm. The outside diameter of theconical apex may be from about 30 μm to about 150 μm.

An energy source, such as a laser or other intense light source, 700 isprovided and provides a beam of energy, indicated by arrow 702, that canbe adjusted or is manufactured to focus to a spot size of a few μm toabout 50 μm. The energy beam 702 is directed to a location, preferablythe center, of the sealed thin membrane 31 and the energy of the beam702 is used to “drill” or “burn” through the membrane 31 that seals theconical apex of the nozzle 50 so that an orifice of the desired diameteris formed and the orifice is in fluid communication with the rest of themicrofluidic channel 30. The spot size of the energy beam 702 can befocused down to a few μm to about 50 μm, preferably down to about 8 μmto about 20 μm. This is shown in FIG. 20 where the energy source 700 isin the form of a laser and the laser beam 702 is centrally focused onthe thin membrane 31. The laser beam can be either pulsed or it can becontinuous. With sufficient power input to ablate the thin membranematerial, an orifice (tip opening) 710 is formed so as to open themicrofluidic channel 30 in the nozzle 50 section, as shown in FIG. 21,thereby creating a functional nozzle 50 where a liquid sample can beconveyed to the outside of the nozzle 50 through the channel 30.

In one embodiment, the energy source 700 is a Nd:YAG laser. In anotherembodiment, the energy source 700 is an excimer laser with a wavelengthof about 248 nm or about 193 nm; and in yet another embodiment, theenergy source 700 can be a CO₂ laser. As previously mentioned, thematerials for injection-molding the conical nozzle 50 can be any numberof suitable polymers, preferably a thermoplastic, or ceramics. Suitableplastics include polyalkanes, such as polypropylene, polyethylenepolystyrene, ethylene-norbornene co-polymer, polymethyl methacrylate,polycarbonate, and others. To achieve a small opening, for example 8 μmdiameter, a short wavelength laser, such as a Nd:YAG or an excimerlaser, is used in combination with a plastic having a good absorptioncoefficient for the laser wavelength used. One skilled in the art oflasers and more particularly, forming channels using lasers, willappreciate the selection of a suitable laser and plastic that isrequired to obtain desirable results.

The figures shown illustrate forming an orifice in one single nozzle 50;however, the same procedure can be repeated for forming plural orificesin an array of nozzles 50. For example, FIG. 22 illustrates a focusedlaser beam 702 depositing energy into the thin sealing membrane 31 ofthe sealed nozzle 50. Since the focused laser beam 702 has a diameterless than the diameter of the sealing membrane, the resulting orificeshown in FIG. 23 has a smaller diameter than the diameter of the orificein FIG. 21 even though the functional nozzles start out with the sealednozzles having identical features. In other words, the diameters of thefocused energy beam can either be substantially equal to the diameter ofthe thin membrane 31 or it can be less than the diameter of the thinmembrane 31. An annular lip 35 is thus formed of ablated thin membranematerial and defines the nozzle opening (second end 34).

The advantages of this aspect of the present invention are that theorifice fabricated at the nozzle tip will likely have a smalldistribution of orifice diameters, i.e., high tolerance of +/−1 μm fromorifice to orifice, and the stringent requirements for the accuracy onthe mold for injection molding, and the control of the molding processwill be relaxed, and functional spray nozzles of a range of orificediameters can be fabricated from injection-molded sealed nozzle partsmade from a single injection mold.

In yet another embodiment shown in FIGS. 24 and 25, the microfluidicdevice is formed such that only the reservoir 60 is formed usinginjection molding techniques and the nozzle 50 itself is a solidstructure as opposed to being partially hollow, as in the otherembodiments. FIG. 24 shows the microfluidic device after being formed byinjection molding as well as showing the energy beam 702 being directedtoward the nozzle tip 52 of the nozzle 50. In this embodiment, theenergy beam 702 serves to not only create the orifice (opening) at thetip portion 52 but also serves to form a nozzle channel 710 that extendsthrough the sold body of the nozzle to the reservoir 60, therebycreating a means for liquid in the reservoir to travel through thenozzle 50 and be discharged therefrom through the orifice formed at thenozzle tip.

While the invention has been particularly shown and described shown anddescribed with reference to preferred embodiments thereof, it will beunderstood by those skilled in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention.

1. A method for manufacturing a microfluidic device comprising a bodyand at least one nozzle integral thereto and extending outwardly fromone face thereof, the body having at least one channel formedtherethrough with a length of the channel being formed though the nozzleand terminating near a sealed apex of the nozzle, the method comprisingthe steps of providing only a single mold which includes a negativeimpression of the channel and the at least one nozzle, the negativeimpression being shaped such that the formed nozzle has a beveled outersurface; injecting a polymeric material into the mold; curing thepolymeric material to form the microfluidic device with the sealed apex;removing the body from the mold; and opening up the sealed apex byforming a nozzle opening by focusing an energy beam from an energysource at the sealed apex such that the focused energy beam is coaxialto the channel in the nozzle until material is ablated at the apex,thereby opening up the channel to the exterior, wherein a diameter ofthe energy beam can be varied to form different shaped and differentsized nozzle openings using the same single mold, thereby permittingdifferent final nozzle constructions to be formed from the same formedmicrofluidic device that has the sealed apex.
 2. The method of claim 1,wherein the step of providing a mold including the step of: forming thenegative impression of the channel such that at least the length of thechannel that is formed in the nozzle has a tapered shaped.
 3. The methodof claim 1, further including the step of: forming the mold such thatthe resulting nozzle opening has a diameter equal to or less than 100 μmand an outside diameter of the nozzle is equal to or less than 150 μm.4. The method of claim 1, further including the step of: forming themold such that the resulting nozzle opening has a diameter equal to orless than 50 μm and an outside diameter of the nozzle is equal to orless than 100 μm.
 5. The method of claim 1, further including the stepof: forming the negative impression of the mold such that the resultingmicrofluidic device has a conically shaped nozzle.
 6. The method ofclaim 1, wherein the diameter of the energy that is focused issubstantially equal to a diameter of a thin membrane that extendsbetween an outer wall of the nozzle and seals the apex of the nozzle. 7.The method of claim 1, wherein the diameter of the energy that isfocused is less than a diameter of the thin membrane that extendsbetween an outer wall of the nozzle and defines the sealed apex,resulting in the nozzle opening being defined by an annular lip formedof the thin membrane.